CN101129463B - Method of preparing red sage root injection - Google Patents
Method of preparing red sage root injection Download PDFInfo
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- CN101129463B CN101129463B CN2007101433062A CN200710143306A CN101129463B CN 101129463 B CN101129463 B CN 101129463B CN 2007101433062 A CN2007101433062 A CN 2007101433062A CN 200710143306 A CN200710143306 A CN 200710143306A CN 101129463 B CN101129463 B CN 101129463B
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Abstract
The invention discloses a preparation method of radix salvia miltiorrhizae injection and quality controlling method, which is characterized by the following: controlling the condensing temperature in the controlling technical course based on traditional technique; utilizing soluble salviol acid as main effective component of tanner and salvia miltiorrhizae to remove residual tanner according to the differential molecular weight. The radix salvia miltiorrhizae injection obtained by the inventive preparation method simultaneously contains four effective components of alviol acid A, alviol acid B, sodium tanshinone and protocatechualdehyde. The content is stable and tannin detection is qualified. The preparation method effectively raises the quality of the radix salvia miltiorrhizae injection, so that the radix salvia miltiorrhizae injection reaches in deed a complete new level with safety, efficiency and controllable quality.
Description
Technical field
The present invention relates to a kind of preparation method of Chinese medicine preparation preparation method, particularly a kind of Radix Salviae Miltiorrhizae Injection.
Background technology
In recent years, along with the raising of China's living standards of the people, the prolongation of life expectancy and the change of diet structure, evidence of coronary heart diseases and mortality rate are just presenting ascendant trend.China has 4,000 ten thousand patients with coronary heart disease at present approximately, per hour has 260 routine patients to die from cardiovascular and cerebrovascular disease approximately.Incidence of CHD is the trend of rapid rising in recent years in China.World Health Organization (WHO) has report to point out that even just take strong preventive measure from now on, the ascendant trend of China's cardiovascular disease incidence rate also will last till about the year two thousand twenty.The rank of cardiovascular and cerebrovascular vessel medication in drug market also rises to second, is only second to the infection medication.It is reported that the total general layout of present domestic cardio-cerebralvascular pharmaceutical market is that Chinese medicine and western medicine respectively accounts for half.Administration data statistics from hospital, the cardio-cerebralvascular preparation is at present still by the leading market of injection, and clinical practice proves, Chinese medicine treatment cardiovascular and cerebrovascular disease has the irreplaceable unique effects of chemical medicine product, the advantage that also has other dosage forms of Chinese medicine to be difficult to possess.
Radix Salviae Miltiorrhizae is as the blood-activating and stasis-removing of classics, be widely used in cardiovascular and cerebrovascular diseases such as treatment coronary heart disease, angina pectoris, ischemia apoplexy, about 5,000,000,000 yuans of red sage formulation annual sales amount, especially Radix Salviae Miltiorrhizae Injection and FUFANG DANSHEN ZHUSHEYE, year sale is up to 3,000,000,000.
Existing Radix Salviae Miltiorrhizae Injection is included in the 20th p34 of Chinese traditional patent formulation preparation promulgated by the ministries or commissions of the Central Government, and standard is numbered: WS3-B-3766-98.Function is a blood circulation promoting and blood stasis dispelling with curing mainly, freeing vessels and nourishing heart.Be used for coronary heart disease, angina pectoris.
Usage and consumption are intramuscular injection, a 2～4ml, 1～2 time on the one; Intravenous injection, a 4ml (using), 1～2 time on the one with 50% glucose injection 20ml dilution back; Intravenous drip, a 10～20ml (using), 1 time on the one with 5% glucose injection, 100～500ml dilution back.Or follow the doctor's advice.
Only with the index components of protocatechualdehyde, contain protocatechualdehyde (C7H6O3) with the every 1ml of its content of high effective liquid chromatography for measuring and must not be less than 0.2mg in the present quality standard promulgated by the ministries or commissions of the Central Government as quality control.The Radix Salviae Miltiorrhizae Injection method for making that this standard is included is: gets Radix Salviae Miltiorrhizae 1500g, decocts with water three times, and 2 hours for the first time, second and third time each 1.5 hours, collecting decoction filters, and filtrate decompression is concentrated into 750ml.Add the ethanol precipitation secondary, make for the first time that to contain the alcohol amount be 75%, make that to contain the alcohol amount be 85% for the second time, each all cold preservation is placed the back and is filtered, filtrate recycling ethanol, and be concentrated into about 250ml, add the injection water to 400ml, mixing, cold preservation is placed, filter, regulate pH value to 6.8, boil half an hour with 10% sodium hydroxide solution, filter, add the injection water to 1000ml, embedding, sterilization, promptly.
Because historical reasons, also there is the problem of quality and clinical safety aspect in present Danshen root injection.It is extract through the comprehensive composition of Chinese crude drug of traditional handicraft preparation that the problem of quality aspect mainly shows as present injection of danshen liquid product, because the not in place and method for making of understanding, quality control method imperfection, exist effective ingredient indeterminate, the quality instability, show as the clinical efficacy instability, the safety poor controllability, the generation untoward reaction is many and be difficult to search reason, is difficult to adapt to the modernization of Chinese medicine and international requirement.
Non-quantitation composition unknown in the present Radix Salviae Miltiorrhizae Injection accounts for suitable ratio; Some macromole impurity are difficult to remove as tannin etc., phenomenons such as color and luster deepens, muddiness, precipitation can occur after placing a period of time, thereby influence the curative effect and the safety of preparation.Tannin is the principal element that influences clarity of injection, and tannin is a relative molecular weight greater than the water solublity polyphenol compound of 1000 (general 3000-5000), and it can same alkaloid, the metal ion effect in protein and the medicine generates precipitation.For example, it and protein molecule, alkaloid and polysaccharide form intermolecular hydrogen bonding, generate water-fast precipitate; Therefore tannin contained in the injection must be removed.In general, tannin enters and very easily causes jaundice and hepatic necrosis in the body, and intramuscular injection is local can harden, pain, and tannin is the derivant of polyhydric phenols, not only has been dissolved in alcohol but also water-soluble.
It is poor that secondary alcohol precipitation process technology is removed the tannin technique effect in the existing ministry standard, and it is qualified that products obtained therefrom is difficult to, and poor selectivity, and the mass efficient composition is removed simultaneously.Existing technology comprises that also cold method, water alcohol method, glue alcohol method, polyamide method and the glue alcohol resin method etc. of putting of heat treatment (see Wang Ling etc. for details, West China pharmaceutical journal, 2003, No.18, p275; Wang Jian etc., basic unit's Chinese medicine magazine, 2001, No.5, p51); Water alcohol method is in order to remove tannin rate height, and general pH value is heightened to 9-10, if under high PH, and a large amount of precipitated removing of salvianolic acid composition; Under the situation of only considering protocatechualdehyde, glue alcohol resin method is had an optimistic view of the most, but in fact owing to the structural similarity of tannin and salvianolic acid constituents, macroporous resin has been taken away a large amount of main effective ingredient.Above-mentioned these are removed the method poor selectivity of tannin, and when removing tannin, a large amount of salvianolic acid effective constituents are removed; Some method is also introduced other impurity, and the quality of product is reduced.
Along with deepening continuously to red sage root component understanding, discovering in recent years, be that water solublity salvianolic acid constituents (general relative molecular weight less than the 1000) cardiac vascular activity of representative is strong more than danshensu, protocatechualdehyde with salvianolic acid B and salvianolic acid A in the Radix Salviae Miltiorrhizae, the salvia-soluble liposoluble ingredient is that the main pharmacodynamics material base of Radix Salviae Miltiorrhizae Injection has obtained academia and generally acknowledges.The salvianolic acid material has extensively and significant pharmacologically active cardiovascular system; have extremely strong antioxidation, can protect myocardial damage, anticoagulant, microcirculation improvement, the promotion vasodilation that ischemia and ischemia-reperfusion cause and improve hemorheology.
What attract people's attention is, critical process point in the Radix Salviae Miltiorrhizae Injection production technology in the existing ministry standard is not stipulated details, link such as particularly concentrating does not control temperature, because main effective ingredient is the water soluble ingredient of thermal sensitivity in the Radix Salviae Miltiorrhizae, these compositions are less stable all, particularly the destruction of salvianolic acid A, salvianolic acid B is serious to effective ingredient for original whole production method, and salvianolic acid A, salvianolic acid B loss are very big, requires further improvement and improves production technology.Studies show that, all easily degraded is unstable in solution for salvia-soluble effective ingredient such as salvianolic acid B, people such as Zhang Jun (CHINA JOURNAL OF CHINESE MATERIA MEDICA vol.30, No.10 p789) once studied the salvianolic acid B stability in the Radix Salviae Miltiorrhizae water extract, and the result shows, 100 ℃ of heating of Radix Salviae Miltiorrhizae water liquid 1 hour, it is about 30% that content of danshinolic acid B reduces, and was heated 9 hours, and content of danshinolic acid B reduces about 95%; 60 ℃ of heating 1 hour, it is about 15% that content of danshinolic acid B reduces, and was heated 9 hours, and content of danshinolic acid B reduces about 60%; 50 ℃ of heating 1 hour, it is about 30% that content of danshinolic acid B reduces, and was heated 8 hours, and content of danshinolic acid B reduces about 40%.People such as other Qu Guiwu (Chinese herbal medicine, 2005, No.11 p1654) discovers, the chemical property of salvianolic acid B is very unstable.Find in the experiment in the aqueous solution salvianolic acid B low quality concentration (＜0.1mg/mL) the ambient temperature overnight loss is near 30% down, its catabolite is danshensu, protocatechualdehyde etc.People such as Yue Xidian (Chinese herbal medicine, 2005, No.2 p205) studies show that, keep salvianolic acid B stability in the solution, should accomplish low solution pH value, high salvianolic acid B mass concentration, low temperature as far as possible, and will shorten the resting period of solution state as far as possible.
Membrane separation technique begins to be applied to the production of Chinese medicine preparation since the eighties.Though this technology is started late, the application scale is less, has received people's very big concern.Wherein the application report with ultrafilter membrane is maximum especially.Relate generally to the following aspects: the 1) extraction of effective ingredient in Chinese; 2) production of oral liquid; 3) preparation of extractum preparation; 4) removal of thermal source; 5) from pharmacy waste water, reclaim effective ingredient.(Chinese herbal medicine, 1996, No.8 such as Hao Li, p471) once carried out the process modification that remove impurity is applied to Radix Salviae Miltiorrhizae Injection with the ultrafilter membrane of molecular cut off 20,000, because used membrane aperture is too big, though can remove the part tannin, relatively poor to the tannin removal effect of relative molecular weight below 5000.The simple simultaneously ultrafiltration removal tannin of using, because content of tannin is higher, the ultrafiltration inefficiency is simultaneously because effects such as absorption cause effective ingredient to lose in a large number.
In the manufacturer of ultrafilter membrane, have to mention KOCH company, the said firm is in (the inventor of hollow-fibre membrane in the world of purchase U.S. Romicon company in 1991, the hollow-fibre membrane ultrafilter membrane has been invented by the said firm in 1973), the hollow-fibre membrane of Romicon company has extremely strong resistance tocrocking, has globally unique molecular weight cut off minimum, the most accurate 1000 molecular weight films.The Romicon hollow-fibre membrane is made by asymmetric man-made polymer, inner surface is one deck ultrafilter membrane as thin as a wafer, this particular performances makes the filter membrane fiber oneself to support and to allow backwash, this asymmetric taper structure also makes pollutant can not be trapped in the pollution of the inner formation of film deep layer, so the class hollow-fibre membrane has stronger antifouling property and cleaning is easily restorative, is widely used in feed separation and wastewater treatment.The hollow-fibre membrane product that KOCH provides has: 1000 molecular weight, 2000 molecular weight, 5000 molecular weight, 10,000,30,000,50,000,100,000,500,000 molecular weight etc.
Gel chromatography technology is a kind of emerging technology, its principle is to utilize the difference of separated material molecular weight size, shape to cause that the penetration degree difference makes component separate (molecular sieve principle) on filler, macromolecular substances is because diffusion hindered, conduct elutes at first than weak point in gel, the small-molecule substance that enters in the gel is eluted from large to small by molecular size, thereby reach the purpose that is separated from each other, character per sample select for use water, buffer solution with salt or organic solvent as mobile phase.Use dextran gel filtration medium Sephadex G10, G15, G25, G50 etc. to remove macromole, the efficient height, treating capacity can reach bed volume 30%.Only need behind sample introduction, to collect the eluent (initial eluent is a macromolecular substances, and is discarded) that begins from the 1/3-1/2 column volume, just can obtain the following micromolecule of this filler separating ranges upper limit, directly simple.Owing to just remove macromole, the high 10cm of post is above to get final product.
| ? | Main performance separating ranges (relative molecular weight) | Model |
| Sephadex G 10 | ＜700 | SephadexG-10 |
| Sephadex G 15 | ＜1500 | SephadexG-15 |
| Sephadex G 25 | 1000-5000 | SephadexG-25 |
In view of in the existing Radix Salviae Miltiorrhizae Injection quality standard only with the index components of protocatechualdehyde as quality control, lagged far behind red sage formulation actual mass control needs.In recent years, in order to improve Quality Control level to the effective ingredient of Radix Salviae Miltiorrhizae Injection, many scholars and mechanism have carried out unremitting research, people such as Pan Haini have set up a kind of quality control evaluation and analytical method of Radix Salviae Miltiorrhizae Injection to being that quality control index is studied with danshensu sodium, protocatechualdehyde, rosmarinic acid and salvianolic acid B; National departments concerned also is about to carry out a new Radix Salviae Miltiorrhizae Injection quality standard in conjunction with fingerprint pattern technology, main quality control index mainly contains 2, the one, the chromatographic fingerprints of Chinese materia medica similarity evaluation system evaluation that can provide by state-promulgated pharmacopoeia, this product finger printing and reference fingerprint compare, and similarity must not be lower than 0.80; The 2nd, the every 1ml of this product contains danshensu sodium (C
9H
9O
5Na) must not be less than 0.60mg; Contain protocatechualdehyde (C
7H
6O
3) must not be less than 0.20mg; Contain salvianolic acid B (C
36H
30O
16) must not be less than 0.25mg.
Further discovering in recent years, in present known water solublity salvianolic acid constituents, salvianolic acid A is wherein the strongest effective ingredient, its drug action is about 12 times of present main flow red sage formulation Quality Control effective ingredient salvianolic acid B.Be difficult to break through owing to obtain the technology of high-load salvianolic acid A for a long time, salvianolic acid A is failed as the quality control index of red sage formulation always always.
To sum up, the major defect of present existing Radix Salviae Miltiorrhizae Injection production technology is that method for making is not quite reasonable, causes main effective ingredient salvianolic acid A, B to lose very big; Shortage causes product clinical efficacy instability to the quality control of crucial effective ingredient salvianolic acid A; Tannin is removed not exclusively, and clinical practice has bigger potential safety hazard.
Summary of the invention
In view of in defective in the preparation method of existing Radix Salviae Miltiorrhizae Injection and the quality standard only with the index components of protocatechualdehyde as quality control, can not adapt to present quality control and the effective requirement of clinical drug safety far away to the Radix Salviae Miltiorrhizae Injection effective ingredient, we are after the production technology (seeing Chinese patent application 200610165779.8 for details) that obtains the high-load salvianolic acid A, by continuous effort, a kind of preparation method and method of quality control thereof of Radix Salviae Miltiorrhizae Injection have been invented, promptly control the thickening temperature in the process engineering, behind the preliminary alkali deposited of low basicity, utilize the difference of tannin (relative molecular weight greater than 1000, general 3000-5000) and the main effective ingredient water solublity of Radix Salviae Miltiorrhizae salvianolic acid constituents (generally relative molecular weight is less than 1000) relative molecular weight to remove the small amount of residual tannin; Adopt the compound tannin technology (deepfreeze that removes of the present invention, low basicity precipitate with ethanol, ultrafiltration or the compound tannin-removing of glucosan sephadex chromatography technology), the main effective ingredient that under the situation of effectively removing tannin, has kept Radix Salviae Miltiorrhizae to greatest extent, adopt and control salvianolic acid A simultaneously, salvianolic acid B, danshensu sodium and protocatechualdehyde (do not have the salvianolic acid constituents strong though studies show that danshensu and protocatechualdehyde effect, but also there is synergism, so these 2 compositions also need Quality Control) effective content be the quality that quality control index has effectively promoted Radix Salviae Miltiorrhizae Injection, the Radix Salviae Miltiorrhizae Injection that the present invention is produced has really reached safe and effective quality controllable brand-new level.
The invention discloses a kind of preparation method and method of quality control thereof of new Radix Salviae Miltiorrhizae Injection.
The preparation method of Radix Salviae Miltiorrhizae Injection of the present invention comprises the steps:
(1) decoct: get Radix Salviae Miltiorrhizae 1500g, decoct with water three times, 2 hours for the first time, second and third time each 1.5 hours fried in shallow oil to finish at every turn and emitted medical filtration (60 mesh sieve), merging filtrate.
(2) concentrate: filtrate is lower than 60 ℃ is evaporated to 750ml, cooling.
(3) precipitate with ethanol: after the concentrated solution cooling, add ethanol earlier, stir, left standstill about 1 hour to containing alcohol amount 75%.Get supernatant, cold preservation 20-48 hour, the icebox temperature was controlled at below 5 ℃.The cold preservation after-filtration is lower than 60 ℃ of distilling under reduced pressure and reclaims ethanol.Reclaim ethanol to the about 300ml of concentrated solution, put and be chilled to room temperature.
(4) add ethanol again to containing alcohol amount 85%, regulate pH value to 8.0, fully stir, left standstill about 1 hour with 40% sodium hydroxide solution.Cold preservation 40-68 hour, the secondary alcohol deposit fluid after the cold preservation was filtered, and was lower than 60 ℃ of distilling under reduced pressure and reclaimed ethanol.Reclaim ethanol to about 250ml (every 1ml contains crude drug in whole 8.5-10g approximately).
(5) ultrafiltration: add the injection water to 400ml, add 10% hydrochloric acid and transfer pH to 6.8, mixing, cold preservation was placed 36-60 hour, adopted the ultrafilter membrane ultrafiltration, collected filtrate.
(6) add an amount of water for injection, heated and boiled 30 minutes is put and is chilled to 60 ℃, adds an amount of 0.1% active carbon, be incubated 10 minutes, filter, taking liquid records original pH value, NaOH solution with 10% is transferred PH to 6.8, complements to 1000ml with the water for injection after filtering, and stirs.Embedding, sterilization, promptly.
Different with traditional Radix Salviae Miltiorrhizae Injection production technology is that the present invention has all carried out clearly limiting to thickening temperature in step (2), (3), (4), is no more than 60 ℃; This temperature is selected 60 ℃ rather than 50 ℃, mainly is that 50 ℃ of required concentration times are oversize, is unfavorable for actual production, and studies show that under the concentrating under reduced pressure state, and to reduce the degree basic difference little for content of danshinolic acid B when 50 ℃ or 60 ℃.
PH value during production craft step of the present invention (4) secondary precipitate with ethanol is 8.0, mainly studies show that, and when pH is high again, the method poor selectivity, when removing tannin, a large amount of salvianolic acid effective constituents are removed; Then do not have the effect of removing tannin when pH value is too low;
The ultrafilter membrane that production craft step of the present invention (5) is used is a hollow-fibre membrane of holding back relative molecular weight 1000-1 ten thousand, for example the Romicon hollow-fibre membrane of KOCH company.The hollow-fibre membrane product that KOCH provides has: 1000 molecular weight, 2000 molecular weight, 5000 molecular weight, 10,000 etc.Because ultrafiltration belongs to the cross flow filter mode, will use the hollow-fibre membrane more slightly larger during actual the use, otherwise filter pressure is excessive than theoretical aperture, effective ingredient loses in a large number simultaneously, influences the ultrafiltration effect, preferred 2000 molecular weight, 5000 molecular weight ultrafilter membranes.
Studies show that step of the present invention (5) can also be to utilize the sephadex chromatography technology generation for hyperfiltration technique.Dextran gel filtration medium of the present invention is Sephadex G10, G15, G25 etc. preferably.
The method of quality control of Radix Salviae Miltiorrhizae Injection of the present invention comprises following content: it comprises carries out qualitative and quantitative control to danshensu sodium, protocatechualdehyde, salvianolic acid B, salvianolic acid A, test test liquid liquid phase collection of illustrative plates under certain chromatographic condition, this product requires every 1ml to contain danshensu sodium (C
9H
9O
5Na) must not be less than 0.65mg; Contain protocatechualdehyde (C
7H
6O
3) must not be less than 0.23mg; Contain salvianolic acid B (C
36H
30O
16) must not be less than 0.28mg, contain salvianolic acid A (C
26H
22O
10) (the new standard of preparing to carry out of country is only controlled following 3 quality control indexs: every 1ml contains danshensu sodium (C must not to be less than 0.10mg
9H
9O
5Na) must not be less than 0.60mg; Contain protocatechualdehyde (C
7H
6O
3) must not be less than 0.20mg; Contain salvianolic acid B (C
36H
30O
16) must not be less than 0.25mg).
Use the present invention, we check the content requirement of above-mentioned 4 quality control indexs as product export check quality control standard, the production testing quality control that the new production process and the detection technique of Radix Salviae Miltiorrhizae Injection is successfully applied to Radix Salviae Miltiorrhizae Injection, owing to increase the strongest salvianolic acid A of salvianolic acid apoplexy due to endogenous wind drug effect newly as quality control index, compare with traditional handicraft and Quality Control technology, gained Radix Salviae Miltiorrhizae Injection product quality is more reliable and stable, the effectiveness and the safety of product solve substantially, really reach safe and effective, reliable in quality.
Quantitative control methodin of the present invention is measured 4 kinds of water soluble ingredient danshensu sodiums, protocatechualdehyde, salvianolic acid B and salvianolic acid As in the Radix Salviae Miltiorrhizae Injection simultaneously for using the gradient elution program to use reversed phase high-performance liquid chromatography; Certain chromatographic condition used in the present invention, comprise following factor: chromatographic column: Agilent 1100 highly effective liquid phase chromatographic systems, Agilent ZORBAX 80A Extend-C18 post (4.6 * 250mm), mobile phase A: water-acetonitrile-formic acid (90: 10: 0.4, v/v), Mobile phase B: acetonitrile; Flow velocity: 1.0mL/min, detect wavelength: 280nm, column temperature: 30 ℃, DAD sweep limits: 190 ~ 400nm.Used linear gradient elution program, actual conditions such as following table 1: table 1: linear gradient elution program
It is an amount of that danshensu sodium, protocatechualdehyde, salvianolic acid B and salvianolic acid A reference substance are got in the preparation of reference substance solution, the accurate title, decide, add 5% methanol and make the mixed solution that every 1ml contains danshensu sodium 0.32mg, protocatechualdehyde 0.10mg, salvianolic acid B 0.12mg and salvianolic acid A 0.05mg, promptly.
The preparation precision of need testing solution is measured this product 2ml, puts in the 10ml measuring bottle, is diluted with water to scale, shakes up, promptly.
Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
The standard reference material HPLC collection of illustrative plates of 4 content controlling index of quality control of the present invention is made up of 4 characteristic peaks as shown in Figure 1.The retention time t of described four characteristic peaks
RFor from left to right being followed successively by: danshensu sodium t
R=5.40min, protocatechualdehyde t
R=9.60min, salvianolic acid B t
R=30.10min, salvianolic acid A t
R=34.30min.
The inventive method can once be measured a plurality of effective ingredient in the Radix Salviae Miltiorrhizae Injection simultaneously.By quantitative control, can improve the safety and the effectiveness of Radix Salviae Miltiorrhizae Injection greatly to main effective ingredient in these 4 Radix Salviae Miltiorrhizaes.The Radix Salviae Miltiorrhizae Injection quality of the pharmaceutical preparations of patent production of the present invention is stable, controllability is strong, and detection method advanced person, accurate, easy, is applicable to the conventional tracking quality check of producing, and the quality of controlling Radix Salviae Miltiorrhizae Injection is played an important role.
The Radix Salviae Miltiorrhizae Injection analytical method is set up and quality stability evaluation situation simply gathers as follows:
Instrument: Agilent1100 type high performance liquid chromatograph, join degasser, quaternary gradient pump, automatic sampler, column oven, diode array detector, UV-detector (Agilent company).
Reagent: acetonitrile is chromatographically pure (a MERCK company), and all the other reagent are analytical pure, and water is pure water.Reference substance: danshensu sodium (middle inspection institute, lot number: 110855-200507), protocatechualdehyde (middle inspection institute, lot number: 110809-200503), salvianolic acid B (middle inspection institute, lot number: 111562-200609), salvianolic acid A (self-control, normalization demarcate content 98.8%).
Chromatographic condition: chromatographic column: Agilent 1100 highly effective liquid phase chromatographic systems, and Agilent ZORBAX 80AExtend-C18 post (4.6 * 250mm), mobile phase A: water-acetonitrile-formic acid (90: 10: 0.4, v/v), Mobile phase B: acetonitrile; Flow velocity: 1.0mL/min, detect wavelength: 280nm, column temperature: 30 ℃, DAD sweep limits: 190 ~ 400nm.Used linear gradient elution program: 0min 0%B, 20min 15%B, 50min25%B, 70min 85%B, 80min 85B%.
Separation condition and the selection that detects wavelength:
Under above-mentioned chromatographic condition, 4 quantitative target composition chromatographic peaks and adjacent peak all reach baseline separation in the Radix Salviae Miltiorrhizae Injection, the peak shape symmetry, and retention time is suitable, the t of danshensu sodium, protocatechualdehyde, salvianolic acid B and salvianolic acid A
RBe respectively: danshensu sodium t
R=5.40min, protocatechualdehyde t
R=9.60min, salvianolic acid B t
R=30.10min, salvianolic acid A t
R=34.30min determines that the sample detection wavelength is 280nm.
Linear relationship is investigated: the above-mentioned reference substance solution 5 μ l of accurate absorption, inject chromatograph of liquid, and measure by aforementioned chromatographic condition.With reference substance solution mass concentration X (g/L) is abscissa, and peak area Y is a vertical coordinate, and the drawing standard curve (sees Table 2: the linear relationship table).Each index components is in quality measurement concentration, and linear relationship is good.Table 2: linear relationship table
| Main component | Regression equation | r | The range of linearity (g/L) |
| Danshensu sodium | Y＝146.9X-474.2 | 0.9994 | 0.64-5.76 |
| Protocatechualdehyde | Y＝22702.0X+270.2 | 0.9999 | 0.48-4.32 |
| Salvianolic acid B | Y＝4039.3X-445.8 | 0.9995 | 0.20-1.80 |
| Salvianolic acid A | Y＝579.5X-294.1 | 0.9994 | 0.01-0.72 |
The precision test:
Instrument precision: the above-mentioned mixing reference substance solution 5 μ l of accurate absorption, inject chromatograph of liquid, measure by aforementioned chromatographic condition.Continuous sample introduction 5 times, each index components relative standard deviation RSD is 0.88-1.47% with calculated by peak area.
Method precision: get 5 parts of test liquids of the parallel preparation of injection of danshen liquid formulation, each test liquid continuous sample introduction is measured 3 times, and each index components relative standard deviation RSD is 1.08-1.46% with calculated by peak area.
Day to day precision: get with a collection of Radix Salviae Miltiorrhizae Injection sample, surveyed its content in continuous 6 days, as a result relative standard deviation RSD all＜1.5%, the result shows that this method day to day precision amount meets the requirements.
Sample stability:
Get the Radix Salviae Miltiorrhizae Injection test sample, respectively at 0hr, 1hr, 2hr, 3hr, 4hr, measure need testing solution danshensu sodium, protocatechualdehyde, salvianolic acid B and salvianolic acid A content by aforementioned chromatographic condition, as a result relative standard deviation RSD all＜1.5%, the result shows that test sample is stable in 4 hours.
Recovery test:
The accurate Radix Salviae Miltiorrhizae Injection 5ml that draws known content, put in the 10ml measuring bottle, it is an amount of to add each reference substance respectively, each reference substance is all joined 3 concentration, each reference substance addition is followed successively by: danshensu sodium (3.9mg, 3.3mg, 2.6mg), protocatechualdehyde (1.4mg, 1.2mg, 0.9mg), salvianolic acid B (1.7mg, 1.4mg, 1.1mg) and salvianolic acid A (0.6mg, 0.5mg, 0.4mg) reference substance solution 1ml, make need testing solution, measure by above-mentioned liquid phase chromatogram condition, the average recovery rate that calculates each Quality Control composition is followed successively by danshensu sodium 99.6% (RSD1.50%), protocatechualdehyde 100.1% (RSD1.37%), salvianolic acid B 99.8% (RSD1.46%), salvianolic acid A 99.5% (RSD1.41%).
Through above-mentioned research, the present invention has determined following chromatographic condition: chromatographic column: Agilent 1100 highly effective liquid phase chromatographic systems, Agilent ZORBAX 80A Extend-C18 post (4.6 * 250mm), mobile phase A: water-acetonitrile-formic acid (90: 10: 0.4, v/v), Mobile phase B: acetonitrile; Flow velocity: 1.0mL/min, detect wavelength: 280nm, column temperature: 30 ℃, DAD sweep limits: 190 ~ 400nm.Used linear gradient elution program: 0min 0%B, 20min 15%B, 50min 25%B, 70min 85%B, 80min 85B%.
Description of drawings
Use the instrument type: high performance liquid chromatograph device model: Agilent 1100
Mobile phase A: water-acetonitrile-formic acid (90: 10: 0.4, v/v), Mobile phase B: acetonitrile;
The gradient mode: 0min 0%B, 20min 15%B, 50min 25%B, 70min 85%B, 80min 85B%,
Detector: diode array (DAD) detector detects wavelength: 280nm column temperature: 30 ℃
The post model: ZORBAX 80A Extend-C18 post (4.6 * 250mm) flow velocitys: 1.0mL/min,
Integration method: area normalization method
The hybrid standard reference substance HPLC collection of illustrative plates of Figure 14 content controlling index
The Radix Salviae Miltiorrhizae Injection HPLC collection of illustrative plates of Fig. 2 embodiment 1 (traditional handicraft) preparation
Fig. 3 embodiment 1 (traditional handicraft, pH=9.0) Zhi Bei Radix Salviae Miltiorrhizae Injection HPLC collection of illustrative plates
Fig. 4 embodiment 2 (traditional handicraft, pH=8.0) Zhi Bei Radix Salviae Miltiorrhizae Injection HPLC collection of illustrative plates
The Radix Salviae Miltiorrhizae Injection HPLC collection of illustrative plates of Fig. 5 embodiment 4 (new technology, ultrafiltration, 5000 molecular weight) preparation
The Radix Salviae Miltiorrhizae Injection HPLC collection of illustrative plates of Fig. 6 embodiment 5 (new technology, sephadex G 15) preparation
The specific embodiment
Below in conjunction with the specific embodiment the present invention is further specified.Among the following embodiment method of inspection of 4 quality control indexs being carried out according to content assaying method of the present invention (is that every 1ml contains danshensu sodium and must not be less than 0.65mg; Contain protocatechualdehyde and must not be less than 0.23mg; Contain salvianolic acid B and must not be less than 0.28mg, contain salvianolic acid A and must not be less than 0.10mg), the tannin inspection technique is carried out under 2005 editions pharmacopeia appendix of tannin test basis injection item.
Embodiment 1Traditional handicraft prepares Radix Salviae Miltiorrhizae Injection
Get Radix Salviae Miltiorrhizae 1500g, decoct with water three times, 2 hours for the first time, second and third time each 1.5 hours, collecting decoction filters, and filtrate decompression is concentrated into 750ml.Add the ethanol precipitation secondary, make for the first time that to contain the alcohol amount be 75%, make that to contain the alcohol amount be 85% for the second time, each all cold preservation is placed the back and is filtered, filtrate recycling ethanol, and be concentrated into about 250ml, add the injection water to 400ml, mixing, cold preservation is placed, filter, regulate pH value to 6.8, boil half an hour with 10% sodium hydroxide solution, filter, add the injection water to 1000ml, embedding, sterilization, promptly.
Test by quality control method of the present invention, every 1ml contains danshensu sodium 1.00mg; Contain protocatechualdehyde 0.90mg; Contain salvianolic acid B 0.10mg, contain salvianolic acid A 0.05mg; The tannin check has a large amount of precipitations.The result judges, by quality control method of the present invention, except that danshensu sodium and protocatechualdehyde met the requirements, tannin check and salvianolic acid A, B were all defective.
Embodiment 2Traditional handicraft is in conjunction with alkali deposited (traditional handicraft, pH=9.0) preparation Radix Salviae Miltiorrhizae Injection
Substantially the same manner as Example 1, different is in second time during precipitate with ethanol, regulates pH value to 9.0 with 40% sodium hydroxide solution, fully stirs, and leaves standstill about 1 hour.Cold preservation, the secondary alcohol deposit fluid after the cold preservation is filtered, and ethanol is reclaimed in distilling under reduced pressure.Add the injection water to 400ml, add 1: 1 hydrochloric acid and transfer pH to 6.8, boil half an hour, filter, add the injection water to 1000ml, embedding, sterilization, promptly.
Test by quality control method of the present invention, every 1ml contains danshensu sodium 0.95mg; Contain protocatechualdehyde 0.80mg; Contain salvianolic acid B 0.05mg, contain salvianolic acid A 0.02mg; Tannin check clarification.The result judges that tannin is up to the standards, but by quality control method of the present invention, except that danshensu sodium and protocatechualdehyde meet the requirements (all low than embodiment 1) and salvianolic acid A, B all defective (all low than embodiment 1).
Embodiment 3Traditional handicraft is in conjunction with alkali deposited (traditional handicraft, pH=8.0) preparation Radix Salviae Miltiorrhizae Injection
The same substantially with embodiment 2, different is in second time during precipitate with ethanol, regulates pH value to 8.0 with 40% sodium hydroxide solution, and other are all identical.
Test by quality control method of the present invention, every 1ml contains danshensu sodium 0.85mg; Contain protocatechualdehyde 0.76mg; Contain salvianolic acid B 0.09mg, contain salvianolic acid A 0.04mg; The tannin check has small amount of precipitate.The result judges, by quality control method of the present invention, except that danshensu sodium and protocatechualdehyde met the requirements, tannin check and salvianolic acid A, B were all defective.Compare with embodiment 1,4 main component content are more or less the same.
Embodiment 4Cryoconcentration of the present invention, the compound tannin-removing technology of ultrafiltration (molecular weight 5000)
The same substantially with embodiment 3, different is after extraction concentrate and when reclaiming ethanol control be lower than 60 ℃ and carry out, in addition at the secondary precipitate with ethanol, increase the ultrafiltration tannin-removing behind the pH=8.0 alkali deposited.Used ultrafilter membrane is Romicon hollow-fibre membrane (5000 molecular weight).
Specific as follows:
(1) decoct: get Radix Salviae Miltiorrhizae 1500g, decoct with water three times, 2 hours for the first time, second and third time each 1.5 hours fried in shallow oil to finish at every turn and emitted medical filtration (60 mesh sieve), merging filtrate.
(2) concentrate: filtrate is lower than 60 ℃ is evaporated to 750ml, cooling.
(3) precipitate with ethanol: after the concentrated solution cooling, add ethanol earlier, stir, left standstill about 1 hour to containing alcohol amount 75%.Get supernatant, cold preservation 20-48 hour, the icebox temperature was controlled at below 5 ℃.The cold preservation after-filtration is lower than 60 ℃ of distilling under reduced pressure and reclaims ethanol.Reclaim ethanol to the about 300ml of concentrated solution, put and be chilled to room temperature.
(4) add ethanol again to containing alcohol amount 85%, regulate pH value to 8.0, fully stir, left standstill about 1 hour with 40% sodium hydroxide solution.Cold preservation 40-68 hour, the secondary alcohol deposit fluid after the cold preservation was filtered, and was lower than 60 ℃ of distilling under reduced pressure and reclaimed ethanol.Reclaim ethanol to about 250ml (every 1ml contains crude drug in whole 8.5-10g approximately).
(5) ultrafiltration: add the injection water to 400ml, add 10% hydrochloric acid and transfer pH to 6.8, mixing, cold preservation was placed 36-60 hour, adopted Romicon hollow-fibre membrane (5000 molecular weight) ultrafiltration, collected filtrate.
(6) add an amount of water for injection, heated and boiled 30 minutes is put and is chilled to 60 ℃, adds an amount of 0.1% active carbon, be incubated 10 minutes, filter, taking liquid records original pH value, NaOH solution with 10% is transferred PH to 6.8, complements to 1000ml with the water for injection after filtering, and stirs.Embedding, sterilization, promptly.
Test by quality control method of the present invention, every 1ml contains danshensu sodium 0.70mg; Contain protocatechualdehyde 0.35mg; Contain salvianolic acid B 0.35mg, contain salvianolic acid A 0.15mg; Tannin check clarification.The result judges that by quality control method of the present invention, tannin check and the Quality Control of 4 main component content all meet the requirements.
Embodiment 5Cryoconcentration of the present invention, the compound tannin-removing technology of sephadex chromatography technology
The same substantially with embodiment 4, use the sephadex chromatography technology instead in the step (5) of different is embodiment 4, adopt sephadex G 15, sephadex column on step (4) the gained medicinal liquid, (initial eluent is a macromolecular substances to collect the eluent that begins from the 1/3-1/2 column volume behind the sample introduction, discarded), eluent adopts appropriate amount of purified water.
Test by quality control method of the present invention, every 1ml contains danshensu sodium 0.69mg; Contain protocatechualdehyde 0.36mg; Contain salvianolic acid B 0.32mg, contain salvianolic acid A 0.13mg; Tannin check clarification.The result judges that by quality control method of the present invention, tannin check and the Quality Control of 4 main component content all meet the requirements.But sephadex G 15 treating capacities are less, not as the ultrafiltration of embodiment 4.
The result of embodiment 1-5 gathers as follows, sees table 3 for details:
Table 3: the examination and test of products of embodiment 1-5 is summary sheet as a result
| Danshensu sodium content (mg/ml) | Protocatechualdehyde content (mg/ml) | Salvianolic acid B content (mg/ml) | Salvianolic acid A content (mg/ml) | The tannin check | The result judges (by quality control method of the present invention) | |
| Embodiment 1 | 1.00 | 0.90 | 0.10 | 0.05 | A large amount of precipitations | Except that danshensu sodium and protocatechualdehyde met the requirements, tannin check and salvianolic acid A, B were all defective. |
| Embodiment 2 | 0.95 | 0.80 | 0.05 | 0.02 | Clarification | Except that danshensu sodium and protocatechualdehyde meet the requirements (all low than embodiment 1) and salvianolic acid A, B all defective (all low than embodiment 1). |
| Embodiment 3 | 0.85 | 0.76 | 0.09 | 0.04 | Small amount of precipitate | Except that danshensu sodium and protocatechualdehyde met the requirements, tannin check and salvianolic acid A, B were all defective.Compare with embodiment 1,4 main component content are more or less the same. |
| Embodiment 4 | 0.70 | 0.35 | 0.35 | 0.15 | Clarification | Tannin check and the Quality Control of 4 main component content all meet the requirements. |
| Embodiment 5 | 0.69 | 0.36 | 0.32 | 0.13 | Clarification | Tannin check and the Quality Control of 4 main component content all meet the requirements. |
| Requirement of the present invention | 0.65 | 0.23 | 0.28 | 0.10 | Clarification |
The present invention's concrete technical scheme required for protection is not limited to the concrete combination of the expressed technical scheme of the foregoing description.
Claims (4)
1. the preparation method of a Radix Salviae Miltiorrhizae Injection comprises the steps:
(1) decoct: get Radix Salviae Miltiorrhizae 1500g, decoct with water three times, 2 hours for the first time, second and third time each 1.5 hours fried in shallow oil to finish at every turn and emitted medicinal liquid, filters merging filtrate with 60 mesh sieves;
(2) concentrate: filtrate is lower than 60 ℃ is evaporated to 750ml, cooling;
(3) precipitate with ethanol: after the concentrated solution cooling, add ethanol earlier, stir, left standstill about 1 hour to containing alcohol amount 75%; Get supernatant, cold preservation 20～48 hours, the icebox temperature is controlled at below 5 ℃; The cold preservation after-filtration is lower than 60 ℃ of distilling under reduced pressure and reclaims ethanol; Reclaim ethanol to the about 300ml of concentrated solution, put and be chilled to room temperature;
(4) add ethanol again to containing alcohol amount 85%, regulate pH value to 8.0, fully stir, left standstill about 1 hour with 40% sodium hydroxide solution; Cold preservation 40～68 hours, the secondary alcohol deposit fluid after the cold preservation is filtered, and is lower than 60 ℃ of distilling under reduced pressure and reclaims ethanol; Reclaim ethanol to about 250ml, be equivalent to every 1ml and contain crude drug in whole 8.5～10g approximately;
(5) ultrafiltration: add the injection water to 400ml, add 10% hydrochloric acid and transfer pH to 6.8, mixing, cold preservation was placed 36～60 hours, adopted the ultrafiltration of Romicon5000 ultrafilter membrane, collected filtrate;
(6) add an amount of water for injection, heated and boiled 30 minutes is put and is chilled to 60 ℃, adds an amount of 0.1% active carbon, be incubated 10 minutes, filter, taking liquid records original pH value, sodium hydroxide solution with 10% is transferred PH to 6.8, complements to 1000ml with the water for injection after filtering, and stirs; Embedding, sterilization, promptly.
2. the preparation method of the described a kind of Radix Salviae Miltiorrhizae Injection of claim 1, described preparation method step 5 is replaced hyperfiltration technique with the sephadex chromatography technology generation, adopt sephadex G 15, sephadex column on the step 4 gained medicinal liquid, collect the eluent that begins from the 1/3-1/2 column volume behind the sample introduction, eluent adopts appropriate amount of purified water.
3. the preparation method of claim 1 or 2 described a kind of Radix Salviae Miltiorrhizae Injectiones, the content of danshensu sodium is no less than 0.65mg in every milliliter of the making Radix Salviae Miltiorrhizae Injection; The content of protocatechualdehyde is no less than 0.23mg; The content of salvianolic acid B is no less than 0.28mg, and the content of salvianolic acid A is no less than 0.10mg.
4. the preparation method of claim 1 or 2 described a kind of Radix Salviae Miltiorrhizae Injectiones, the Radix Salviae Miltiorrhizae Injection that makes is according to tannin inspection technique under 2005 editions pharmacopeia appendix injection items, and tannin is up to the standards.
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| CN101278972B (en) * | 2008-05-29 | 2011-05-04 | 山东华信制药集团股份有限公司 | Technique for preparing Salvia miltiorrhiza injection with pH gradient sedimentation method combined with hyperfiltration technique |
| CN101703571B (en) * | 2009-11-13 | 2012-01-25 | 广东药学院 | Detection method of compound injection liquid |
| CN101717384B (en) * | 2009-11-24 | 2011-10-12 | 正大青春宝药业有限公司 | Compound in salvia miltiorrhiza bungered sage root injection and application thereof in curing treating cardiovascular disease |
| CN101985417B (en) * | 2010-11-02 | 2013-06-05 | 贵州景峰注射剂有限公司 | Process for extracting salvianic acid A |
| CN103245687B (en) * | 2013-05-07 | 2017-10-13 | 江苏省中医药研究院 | A kind of test method of quality control based on component structure of danshen injections |
| CN104758352A (en) * | 2014-01-02 | 2015-07-08 | 成都百草和济科技有限公司 | Red sage root injection and preparation method thereof |
| CN105628696A (en) * | 2015-12-28 | 2016-06-01 | 上海景峰制药有限公司 | Method for determining content of total phenolic acid in salivae miltiorrhizae liguspyragine hydrochloride and glucose injection |
| CN115429752B (en) * | 2022-09-21 | 2023-07-18 | 常熟雷允上制药有限公司 | Red sage root injection and its preparation method and application |
| CN116642973B (en) * | 2023-05-29 | 2024-01-12 | 江苏神龙药业有限公司 | Method for evaluating quality uniformity among batches of salvia miltiorrhiza injection |
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